Technology

Overview
The FemtoSmart Galvo is a galvanometric scanner-based two-photon microscope which enables in vivo and in vitro functional imaging to be focused on the region of interest (ROI). The scanner consists of two galvanometer-based motor-driven mirrors, meaning that the focal spot can be positioned where required. The high accuracy and this positioning freedom support flexible approaches for ROI creation. In contrast to sampling all pixels in images, ROI scanning (e.g. along a predefined line) restricts scanning to the regions which are relevant for the scientist, resulting in faster recording, and elimination of background noise. This helps to reveal cellular signaling and action potentials.

Key features

For in vivo functional imaging in deep tissues
Each cell body, axon, dendrite and spine can be measured separately
Flexible imaging modes, patented solutions for fast imaging only on the regions of interest
High signal to noise ratio
Intelligent control software

Advanced 2D scanning methods

The advantages of the galvo scanner combined with the intelligent, user-friendly control software enables the user to use many scanning patterns covering the ROIs distributed across the field of view. These patterns have been developed based on the most frequent requests by neuroscientists. For example, multiple frame scanning focuses on cell bodies, multiple line scanning enables us to follow action potentials along dendrites, and random-access point scanning allows measurement or photo stimulation of subcellular components of the highest temporal resolution. Many features of the software, such as real-time display, analysis functions, ∆F/F calculations and integrated parallel data acquisition of electrical recordings promote greater understanding of the physiological processes under the focus of your research.

Multiple line scanning

Multiple line scanning has been developed for researchers who want to resolve dendritic and even spine activity of neurons approaching near real-time measurement mode. During this scanning mode, the X and Y mirrors direct the laser beam flexible along straight lines or complex curves. The scanner spends most of its time collecting signals from these lines, while the intermediate sections between the lines are skipped. In this way, the scanning speed and the signal-to-noise ratio (SNR) of the signals sampled from the multi-site ROIs increases 3- to 4-fold compared to frame scanning.

Folded frame scanning

This patented method enables imaging of a confined area along a line, where the shape of the selected regions can be straight or curved. This advanced scanning method is useful for imaging single cell bodies in different regions of the specimen, or following events along winding dendrites with their protrusions, even while the tissue is moving.

Photostimulus patterns

Uncaging, optogenetics, and other photo stimulation techniques are also supported by our unique scanning patterns and their combinations. Random-access point scanning can be used for stimulation in femtoliter volumes near dendritic spines where the duration of the stimulation, can be set from microseconds to seconds precisely to the experiment. The evoked signals can be followed along the dendrite by line scanning near simultaneously with the photo stimulation. The microsecond-scale switching time between the stimulation and imaging is achieved by using of a Pockels cell and gated detectors.

High signal-to-noise ratio

Subtle changes in the evoked signals can be revealed because of the following features:

scanning only the relevant part of the field-of-view, and skipping the background, result in a very high signal-to-noise ratio,
photon collection efficiency is enhanced thanks to our patented travelling detector system, which uses the shortest possible optical path,
the most sensitive GaAsP photomultipliers available (quantum efficiency >40%) collect scattered photons.

Software

Overview
FemtoSmart Galvo microscope is controlled and works together with our modular measurement control and analysis software package called MES. MES is written in the MATLAB environment enabling rapid code development and opens the data to the users. It is designed with the day-to-day lab experiences in the field of cellular and network imaging. MES supports 2D ROI selection and immediate ROI activity analysis necessary for high throughput measurements.

Labmaster

MES fully integrates the control of all hardware units in the microscope:
galvo scanner, PMTs, light path actuators, electrical devices,
focusing, camera handling,
auxiliary digital and analog channels to interface stimulators,
behavior control devices,
sample stage or patch pipette devices.

MES stores all metadata of the aboves in a complex file containing multiple measurement units along with your comments.

ROI selection

Numerous software tools support all kinds of 2D scanning pattern generation: points, straight or curved lines and areas with various shapes can be selected improving the efficiency of imaging.

  • LineMagic allows easy placing of predefined shapes, such as spirals,
  • Cell3DFinder locates cells for ROI scanning,
  • TravellingSalesman creates the shortest pathway between the scanning points increasing the velocity of the scanning,
  • ROIs can be imaged in the three-dimensional space by RollerCoaster software and hardware package.

Real-time display and analysis

Integrated, quantitative intensity-based calculations allow following real-time fluorescence changes (e.g. ∆F/F calculation) and simultaneous analysis. To extend the function, MES can adapt electrophysiological recordings too displaying Ca2+ and electrical signals side-by-side.

Batch processing

The analysis module of MES contains efficient tools to analyze entire multi-ROI measurement sets conveniently. Sophisticated curve analysis tool allows fast exploration of the measurement data. Having direct access to the data, any user created analysis methods can be developed.

Extension modules

MES can be extended with many software modules to support various measurement protocols. It involves import and export functions with other programs such as MS Excel, Imaris, Amira, Huygens. It can integrate the control of numerous devices (Luigs&Neumann, PipettePanipulation). MES can adapt electrophysiological recordings.

Versatility

optional modules

 

Applications

Deep brain imaging
Thanks to two-photon laser technology and our optical developments, you can study cell bodies and dendrites at a high spatial resolution down to a depth of 850 µm with no photodamage. The 3D slicer module of the control software implements XZ or YZ sectioning and projection of the Z-stacks and enables 2D visualization of 3D stacks projected to any of the three axes.

Calcium imaging

The fast scanning speed on user-defined, separated regions ensures precise and repeatable measurements of rapid changes in the Ca2+-level of neurons and their dendrites. The ratio-imaging software tool offers algorithms for eliminating background noise, and determinates the relative fluorescence changes, displaying them as transient curves as a function of time. The batch analysis tool contains efficient tools for analyzing entire multi-ROI measurement

Intravital imaging

The infrared excitation laser can penetrate thick specimens, enabling living cell behavior in intact tissues and organs to be visualized at high resolution for extended periods with no phototoxicity: essential for morphological studies. The metaprotocol module of the control software automates these measurements on a timescale of seconds by letting you run freely composed sequences of image acquisition.

Parallel electrophysiology

Hardware and software tools help parallel two-photon imaging and aligned electrophysiological recordings. Precise triggering the external recording ensures time aligned measurement of the electrophysiology and imaging, providing different aspects for studying the neuronal cell and network activity. Software module helps automatic importing recorded traces for parallel analysis. Figure shows calcium imaging and patch-clamp recording in an OGB-1 and Alexa-594 filled hippocampal neuron.

Specification

  • in vivo deep brain imaging down to 850 µm

  • 800 µm × 800 µm FOV (with a 20x obj.)

  • 2D scanning modes

    point and random access point with 200 µs/point
    free hand line and multiple line: 40 lines/5 ms
    frame with 4.1 fps at 512 x 512 pixel, 750 µm x 750 µm
    folded frame, multiple folded frame

  • pixel dwell time adjustable: 0.5 µs - 10 ms, pixel-based averaging

  • minimized optical path length by patented travelling detector system

  • non-descanned, ultrasensitive GaAsP PMT

    (>40% quantum efficiency)

  • high signal-to-noise ratio

  • simultaneous detection of multiple wavelength

  • custom-designed optical elements for maximal transmission efficiency

  • MATLAB-based control software with analysis and upgrade possibilities

  • ∆F/F, ∆G/R calculation

  • parallel recording and analysis of electrophysiological data

  • CMOS camera

  • compatibility with extended IR wavelength range

References

Combined two-photon imaging, electrophysiological, and anatomical investigation of the human neocortex in vitro. Balint Peter Kerekes, Kinga Toth, Attila Kaszas, Balazs Chiovini, Zoltan Szadai, Gergely Szalay, Denes Palfi, Attila Bago, Klaudia Spitzer, Balazs Rozsa, Istvan Ulbert, Lucia Wittner, Neurophotonics (2014)

Enhanced Dendritic Action Potential Backpropagation in Parvalbumin-positive Basket Cells During Sharp Wave Activity. B. Chiovini, GF. Turi, G. Katona, A. Kaszas, F. Erdelyi, G. Szabo, H. Monyer, A. Csakanyi, ES. Vizi, B. Rozsa, Neurochemical Research (2010)

Local Postsynaptic Voltage-Gated Sodium Channel Activation in Dendritic Spines of Olfactory Bulb Granule Cells. Wolfgang G. Bywalez, Dinu Patirniche, Vanessa Rupprecht, Martin Stemmler, Andreas V.M. Herz, Denes Palfi, Balazs Rozsa, Veronica Egger, Neuron (2015)

MRZ-99030 – A novel modulator of Aβ aggregation: II – Reversal of Aβ oligomer-induced deficits in long-term potentiation (LTP) and cognitive performance in rats and mice. Rammes G, Gravius A, Ruitenberg M, Wegener N, Chambon C, Sroka-Saidi K, Jeggo R, Staniaszek L, Spanswick D, O’Hare E, Palmer P, Kim EM, Bywalez W, Egger V, Parsons CG, Neuropharmacology (2015)

Matching Cell Type to Function in Cortical Circuits. Luc Estebanez, Jens Kremkow, James F.A. Poulet, Neuron (2015)

Plasticity of intrinsic excitability in mature granule cells of the dentate gyrus. Jeffrey Lopez-Rojas, Martin Heine & Michael R. Kreutz, Nature (2016)
Plasticity of intrinsic excitability in mature granule cells of the dentate gyrus. Jeffrey Lopez-Rojas, Martin Heine & Michael R. Kreutz, Nature (2016)

FemtoSmart Galvo

Galvanometric scanner-based two-photon microscope which enables in vivo and in vitro functional imaging to be focused on the region of interest (ROI).